The present invention is directed to a molecular motor actin binding protein, designated Nuclear Myosin Ixcex2 (NMIxcex2), that includes a 16 amino acid N-terminal extension not present in in other myosin proteins. The 16 amino acid extension is unique to nuclear myosin Ixcex2 and is responsible for the nuclear localization of this protein.
Myosin I is a member of a superfamily of actin binding proteins that hydrolyze adenosine triphosphate (ATP) (Mermall et al., 1998). Most members of this superfamily have been identified based on their DNA sequences. Myosin II is the prototypic member of this family. Myosin II is a two headed, filamentous protein that is an actin-activated ATPase. Myosin II is widely distributed in eukaryotic cells and its role in energy conversion during muscle contraction is well known.
Myosin I is a single headed, non-filamentous, actin-activated ATPase (Pollard and Korn, 1973). First described in Acanthamoeba castellani (Pollard and Korn, 1973), myosin I is widely distributed in metazoan cells (Mermall et al., 1998). There are at least four different subclasses of myosin I proteins, all containing a 110-150 kD heavy chain and 1-6 light chains located in the neck region between the head and tail. In vertebrate forms of myosin I, this light chain is calmodulin.
Immunofluorescence studies of mammalian cells have shown that myosin I is diffusely distributed throughout the entire cytoplasm and that it concentrates near cortical surfaces and in the perinuclear region. Although evidence of specific roles of myosin protein in metazoan cells is lacking, it has been suggested, based on localization studies, that myosin I proteins are molecular motors involved in plasma membrane extension, vesicle and organelle and mechanochemical regulation of calcium channels in hair cells (Mermall et al., 1998).
The nucleus contains a filamentous network that is analogous to the cytoskeleton, and nascent DNA has been shown to tightly associate with the nuclear xe2x80x9cmatrixxe2x80x9d (Berezney and Coffey, 1975). Although the composition of this matrix or nucleoskeleton is unclear, it has been suggested that replication of DNA occurs as templates move through replication xe2x80x9cfactoriesxe2x80x9d anchored to the nucleoskeleton (Cook, 1991). Transcription of ribosomal genes takes place in specific locations in nucleoli known as fibrillar centers. Templates move through an array of RNA polymerases on the surface of those fibrillar centers that are part of a dynamic model of transcription.
Actin is a protein that is abundant in the nucleus (Rando et al., 2000) and is frequently found in association with the nuclear matrix. Actin is reported to associate with small ribonucleoproteins in the processing and transport of RNA. Nuclear-specific actin binding proteins also have been described. Because the injection of anti-actin antibodies into Xenopus oocytes blocks chromosome condensation and the transcription of lampbrush chromosomes (Scheer et al., 1984), actin may be involved in nuclear movements, transcription or other events that require energy. Actin usually works in concert with myosin. Although the presence of myosin II-like proteins in the nucleus has been suggested, these proteins have not been purified or characterized.
Transcription is a process of constructing messenger RNA (mRNA) molecules using a DNA molecule as a template that results in transfer of genetic information to mRNA. Transcription requires energy, and RNA polymerases have been suggested to power the movement of the transcription complexes in a manner analogous to energy conversion by myosin (Yin et al., 1995). However, the precise mechanism and the molecules involved in energy conversion during transcription are not known.
There is a particular need for cellular molecules or molecular structures that can be used to design target pharmaceuticals, as well as a need for the sequences that encode these molecules. Any protein involved in transcription is a site for therapeutic intervention in proliferative disorders.
An actin-based molecular motor of the myosin superfamily is a candidate as an energy conversion molecule involved in transcription.
The present invention is directed to a protein designated Nuclear Myosin Ixcex2 and the corresponding oligonucleotide sequence. It is a new protein that contains a unique N-terminal sequence, and is a separate gene product that belongs to the myosin Ixcex2 family. The myosin superfamily is composed of at least fourteen members including myosin I and myosin II. The myosin I family includes the myosin Ixcex2 form. Both the known cytoplasmic and the newly discovered nuclear forms of this protein are members of the myosin I subfamily of the myosin superfamily of proteins.
The invention is a myosin-like protein in the nucleus. Special techniques, including the use of a unique antibody and partial denaturation followed by renaturation prior to immunoprecipitation, were used to isolate myosin Ixcex2, a member of the myosin superfamily, from the nucleus and to show that it is required for transcription.
It was unexpectedly discovered that the 120 kD protein is a new isoform of myosin Ixcex2 that has 98% amino acid sequence homology to the previously known cytoplasmic myosin Ixcex2, but its functionality is determined by a newly sequenced characteristic 16 amino acid N-terminal extension that is not found in cytoplasmic myosin I.
There has been no previous suggestion of a myosin Ixcex2 protein containing a 16 amino acid N-terminal extension or that this 16 amino acid extension causes nuclear localization of that protein or that myosin Ixcex2 is included in the transcription process.
The Nuclear Myosin Ixcex2 protein has an amino acid sequence shown in FIG. 1 (GenBank Accession Number AY 007255). This amino acid sequence includes an initiator methionine and a 16 amino acid peptide N-terminal to the initiator methionine. The peptide includes the amino acid sequence MRYR ASAL GSDG VRVT (SED ID NO:2) at the N-terminal end.
In an embodiment of the present invention, a complementary DNA molecule (cDNA) is synthesized from isolated mRNA""s of the Nuclear Myosin Ixcex2 (NMIxcex2) 16 amino acid N-terminal extension (FIG. 2).
In another embodiment of the present invention, antibodies are directed to the Nuclear Myosin Ixcex2 protein. Additionally, antibodies directed to the peptide comprising the amino acid sequence MRYR ASAL GSDG VRVT (SED ID NO:2) are produced. The antibodies directed to Nuclear Myosin Ixcex2 protein or to the 16 amino acid peptide as described herein may also be monoclonal antibodies.
In another embodiment of the present invention, electron and confocal microscopy revealed that Nuclear Myosin Ixcex2 co-localizes with RNA polymerase II. RNA polymerase II and Nuclear Myosin I were also shown to co-precipitate, demonstrating that RNA polymerase II and Nuclear Myosin I bind to each other to form a complex.
In another embodiment of the present invention, the Nuclear Myosin Ixcex2 protein forms a functional complex with RNA polymerase II. This complex is actively involved in the transcription process that can be disrupted by inactivating Nuclear Myosin I. The ability to suppress or disrupt the transcription process is useful in designing treatments and gene therapies for conditions wherein cell proliferation should be inhibited.
In an embodiment of the present invention a method is provided for inhibiting cell proliferation. The method comprises the steps of obtaining antibodies to the 16 amino acid N-terminal extension of the Nuclear Myosin Ixcex2 protein and then administering the antibodies to an organism wherein the antibodies contact cells and may inhibit transcription. The antibodies of this method include monoclonal antibodies.
In another embodiment of the present invention, a screening method for inhibiting transcription is disclosed.
NMIxcex2=nuclear myosin Ixcex2.
CMIxcex2=cytoplasmic myosin Ixcex2 (ie: NMIxcex2 without its characteristic 16 amino acid N-terminal extension).
Molecular motors=molecules that hydrolyze nucleotides and use the energy that is released to do mechanical work at the molecular level. 5xe2x80x2UTR=the untranslated region at the 5 prime end of a nucleotide sequence. 5xe2x80x2RACE=rapid amplification of cDNA ends.
PBS=phosphate buffered saline.
Isoforms=multiple forms of the same protein that differ in their amino acid sequences and are produced by different genes or separate transcripts.
kD=kilodalton.
DTT=dithiothreitol.
EDTA=ethylenediaminetetracetic acid.
EGTA=ethylene glycol-bis(xcex2-amino ethyl ether).
PMSF=phenylmethylsulfonyl fluoride.
BrUTP=5-bromouridine 5xe2x80x2-triphosphate.
ATP=adenosine 5xe2x80x2-triphosphate.
CTP=cytidine 5xe2x80x2-triphosphate.
GTP=guanosine 5xe2x80x2-triphosphate.
UTP=uridine 5xe2x80x2-triphosphate.
The single letter code for amino acids is used herein.